Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro e ither directly by enzyme-induced deamination of cytosine to uracil in the a bsence of the methyl donor, S-adenosylmethionine (SAM), or indirectly throu gh spontaneous deamination of [5-methyl]cytosine to thymine, Using a Lac re version assay, we investigated the contribution of the first mechanism to D cm mutagenesis in vivo by lowering the levels of SARI, Escherichia coli SAR I levels were lowered by reducing SARI synthetase activity via the introduc tion of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SA RI hydrolase, The metK84 strains exhibited increased C-to-T mutagenesis. Ex pression of the T3 SARI hydrolase gene, under the control of the arabinose- inducible P-BAD promoter, effectively reduced Dcm-mediated genomic DNA meth ylation. However, increased mutagenesis was not observed until extremely hi gh arabinose concentrations were used, and genome methylation at Dcm sites was negligible.